Mr. Paul M. Hwang Profile

Paul M. Hwang

at National Institutes of Health

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Proceedings Article | 4 March 2022 Poster + Paper

Fluorescence lifetime imaging of metMyoglobin formation due to nitric oxide stress

Rozhin Penjweini, Mateus Mori, Paul Hwang, Dan Sackett, Jay Knutson

Proceedings Volume 11965, 119650H (2022) https://doi.org/10.1117/12.2608888

KEYWORDS: Fluorescence resonance energy transfer, Hypoxia, Oxygen, Sensors, Fluorescence lifetime imaging, NOx

Read Abstract +

Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe³⁺ state of myoglobin, metmyoglobin (metMb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and metMb proportions- or, more broadly, of the mechanisms by which metMb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O₂) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O₂ occupancy -or upon metMb production control the FRET rate from the fluorescent protein to myoglobin. When O₂ is bound, mCherry fluorescence is only slightly quenched, but if either O₂ is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to metMb with absorbance increases in the red visible range. mCherry thus senses both met and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating metMb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to metMb while being indifferent to O₂ occupancy.

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